Objectives We attempted to compartmentalize the periaqueductal gray (PAG) of the rabbit in terms of the different distribution patterns between NADPH-diaphorase (NADPHd)- and calbindin D28K (CB)-positive neurons. Methods Immunohistochemistry and immunofluorescent labelling for CB and histochemistry for NADPHd were carried out on coronally-sectioned midbrain slices of the rabbit. Results NADPHd-positive neurons were selectively localized in the dorsolateral (DL), the middle one-third of the lateral (L), the dorsal half of the ventrolateral (VLd) PAG, and the supraoculomotor cap nucleus (Su3C). Clusters of CB-immunoreactive perikarya marked the dorsal half of DL (DLd), Su3C, the ventral one-third of L, and the ventral half of the ventrolateral (VLv) PAG. Double labelling for NADPHd and CB revealed that two markers labelled different neuronal groups in DLd and Su3C subdivisions. Conclusion The present data suggest that NADPHd and CB can be regarded as reliable neurochemical markers to reveal the longitudinally-columnar organization within the PAG and to subdivide each columnar area.